An Nuclear Magnetic Resonance Fingerprint Matching Approach for the Identification and Structural Re-Evaluation of Pseudomonas Lipopeptides

ABSTRACT Cyclic lipopeptides (CLiPs) are secondary metabolites secreted by a range of bacterial phyla. CLiPs from Pseudomonas in particular, display diverse structural variations in terms of the number of amino acid residues, macrocycle size, amino acid identity, and stereochemistry (e.g., d- versus l-amino acids). Reports detailing the discovery of novel or already characterized CLiPs from new sources appear regularly in literature. Increasingly, however, the lack of detailed characterization threatens to cause considerable confusion, especially if configurational heterogeneity is present for one or more amino acids. Using Pseudomonas CLiPs from the Bananamide, Orfamide, and Xantholysin groups as test cases, we demonstrate and validate that the combined 1H and 13C Nuclear Magnetic Resonance (NMR) chemical shifts of CLiPs constitute a spectral fingerprint that is sufficiently sensitive to differentiate between possible diastereomers of a particular sequence even when they only differ in a single d/l configuration. Rapid screening, involving simple matching of the NMR fingerprint of a newly isolated CLiP with that of a reference CLiP of known stereochemistry, can then be applied to resolve dead-ends in configurational characterization and avoid the much more cumbersome chemical characterization protocols. Even when the stereochemistry of a particular reference CLiP remains to be established, its spectral fingerprint allows to quickly verify whether a newly isolated CLiP is novel or already present in the reference collection. We show NMR fingerprinting leads to a simple approach for early on dereplication which should become more effective as more fingerprints are collected. To benefit research involving CLiPs, we have made a publicly available data repository accompanied by a ‘knowledge base’ at https://www.rhizoclip.be, where we present an overview of published NMR fingerprint data of characterized CLiPs, together with literature data on the originally determined structures. IMPORTANCE Pseudomonas CLiPs are ubiquitous specialized metabolites, impacting the producer’s lifestyle and interactions with the (a)biotic environment. Consequently, they generate interest for agricultural and clinical applications. Establishing structure-activity relationships as a premise to their development is hindered because full structural characterization including stereochemical information requires labor-intensive analyses, without guarantee for success. Moreover, increasing use of superficial comparison with previously characterized CLiPs introduces or propagates erroneous attributions, clouding further scientific progress. We provide a generally applicable characterization methodology based on matching NMR spectral fingerprints of newly isolated CLiPs to natural and synthetic reference compounds with (un)known stereochemistry. In addition, NMR fingerprinting is shown to provide a suitable basis for structural dereplication. A publicly available reference compound repository promises to facilitate participation of the lipopeptide research community in structural assessment and dereplication of newly isolated CLiPs, which should also support further developments in genome mining for novel CLiPs.


Analyses methods -schematic
: Overview of the analysis steps for the stereochemical validation of CLiPs, as used in this report. With well over 100 distinct Pseudomonas CLiPs reported to date each with their own trivial name, we find it convenient to formally propose a simple and convenient (l: m) notation that will be used throughout to classify structurally homologous CLiPs in particular groups named for a member that represents the group prototype. It is based on 1) the total number of amino acid residues or 'length' l of the peptide sequence and 2) the number of amino acids m contained within the macrocycle, to assist in their classification. Using this, a total of 17 sequences featuring a nonapeptide (l=9) chain with seven residues forming the macrocycle (m=7) and amphipathic profile matching that of viscosin together define the (9:7) Viscosin group. In this way, individual CLiPs or groups of CLiPs introduced over time by different authors such as the PPZPMs and poaeamides can structurally be accommodated in the Orfamide group, all being (10:8) CLiPs with identical amphipathic profile. Similarly, MDN-0066, a CLiP issued from screening Pseudomonas sp. against a cancer cell platform, is a (8:6) CLiP that can be considered as a structural homologue of the Bananamide group.
LC-MS analysis was performed on an Agilent (Santa Clara, CA, U.S.A) 1100 Series HPLC with an ESI detector type VL, equipped with a Kinetex column (C18, 150  4.60 mm, 5 µm particle size) with a flow rate of 1.5 ml/min. Two different solvent systems were used, either 5 mM NH4OAc in H2O (A) and CH3CN (B) or 01% HCOOH in H2O (A) and CH3CN (B). Optimized different gradients were used for each CLiP.
High-resolution mass spectra for all natural CLiPs were recorded on a 6220 A TOF-MS (Agilient Technologies) equipped with an ESI/ASPCI multimode source (Agilient Technologies) set to APCI mode only.
Semi-preparative purification was performed on an Agilent 218 solvent delivery system with a UV-VIS dual wavelength detector using a Phenomenex column (AXIA packed Luna C18(2), 250  21.2 mm, 5 µm particle size) with a flow rate of 17.5 ml/min and following solvent systems: H2O containing 0.1% TFA (A) and CH3CN (B).
Preparative purification of the crude peptides via RP-preparative-HPLC was performed on a Gilson PLC 2250 instrument with a Waters Millipore Corporation DeltaPak C18 PrepPak cartridge (pore size 100 Å and spherical silica of 15 µM) connected to a multiwavelength detector. The solvents used for the measure of samples are H2O (A) and MeCN (B), both containing 0.1%TFA, at a flow rate of 65 mL min -1 .
NMR characterization of intermediate products: 1 H NMR and 13 C NMR were recorded in CDCl3 on a Bruker Avance spectrometer equipped with a 5 mm BBO probe and operating at 300 MHz and 75.77 MHz respectively. High-resolution mass spectra were recorded on an Agilent 6220A time-offlight mass spectrometer, equipped with an Agilent ESI/APCI multimode source. The ionization mode was set to APCI (atmospheric pressure chemical ionization), while the mass spectra were acquired in 4 GHz high-resolution mode with a mass range set to 3200 Da.
NMR characterization of the final compounds were performed either on a Bruker (Billerica, MA, U.S.A) Avance III spectrometer operating at a frequency of 500.13 MHz and 125.76 MHz for 1 H and 13 C respectively and equipped with a 5 mm BBI-Z probe or on a Bruker Avance II spectrometer operating at a frequency of 700.13 MHz and 176.05 MHz ( 1 H and 13 C respectively.) equipped either a 1 H, 13 C, 15 N TXI-Z probe or 5 mm Prodigy TCI probe. All NMR measurements on the final compounds were performed on lipopeptide solutions with 600 µl of CD3CN, DMF-d7, methanol-d3 or DMSO-d7(Eurisotop, Saint-Aubin, France). 1 H and 13 C chemical shift scales were calibrated by using the residual solvent signal. High quality HP-7 (New Era Ent. Inc) NMR tubes were used. Sample temperature was set to 25°C unless stated otherwise. All spectra were processed and analysed using TOPSPIN 4.0.8 and were referenced based on the used solvent.
Full assignment of the peptides was achieved through liquid state NMR spectroscopy using 1D 1 H, 2D 1 H-1 H COSY, 1 H-1 H TOCSY with 90 ms spinlock, 1 H-1 H off-resonance ROESY with a mixing time of 200 ms and gradient-enhanced 1 H-13 C HSQC and 1 H-13 C HMBC optimised for a n JCH coupling of 6.5 Hz. The spectral width was set to 14 ppm in the 1 H dimension and 90 ppm (gHSQC) or 200 ppm (gHMBC) along the 13 C dimension and, generally, 2048 data points were sampled in the direct dimension and 512 data points in the indirect dimension. Standard pulse sequences available in the Bruker library were used throughout with excitation sculpting when applicable. For 2D processing, the spectra were zero filled to a 2048 × 2048 real data matrix and all spectra were multiplied with a squared cosine bell function in both dimensions or a sine bell in the direct dimension for gHMBC before Fourier transformation.

Synthesis of building blocks
The building blocks necessary for the synthesis of the bananamide analogues, orfamide B and xantholysin A, including Fmoc D-Gln(NH2)-OAll, Fmoc-D-Ser(OH)-OAll, Alloc-L-Ile, Alloc-L-Leu and the lipid tail ((R)-3-(tert-butyldimethylsilyloxy)decanoic acid) were synthesized according to the methods described in literature (12). The enantiomeric excess of the lipid tail was determined using chiral HPLC analysis run on a Daicel Chiracel ODH kolom (250x4.6 mm) with an isocratic elution of hexane/EtOH (97/3) over 30 minutes and a flow rate of 1 ml/min. An enantiomeric excess of 99.0% of the (R)-enantiomer was established after the asymmetric hydrogenation step. The obtained NMR spectral data were in accordance with earlier reported data (13).

Solid Phase Peptide Synthesis
Automated peptide synthesis was performed in plastic reaction vessels equipped with Teflon frits (MultiSyn Tech GmbH (Witten, Germany)). The automated peptide synthesis was executed with a SYRO II Multiple Peptide Synthesizer Robot, equipped with a vortex unit, at room temperature. The solid phase peptide synthesis made use of the ubiquitous Fmoc/tBu protecting strategy with HBTU/DIPEA as coupling reagents. The Fmoc deprotection step was performed by treatment with a solution of 40% piperidine in NMP(v/v) for 4 minutes and repeated for another 12 minutes. The coupling step was carried out on the resin using Fmoc protected amino acids (5 equiv.) dissolved in DMF in the presence of HBTU (5 equiv) and DIPEA (10 equiv.) for 40 minutes at room temperature. The coupling step was performed twice to ensure complete coupling of the D-amino acids.
For a manual removal of the Fmoc group, the resin was typically treated with 20% piperidine in DMF (v/v) (10 ml/g resin) for 2 after which the reaction mixture was filtered and the resin was again treated subsequently 10 and 20 minutes with 20% piperidine in DMF. Subsequently, the resin was washed with DMF, MeOH and DCM (610s, 10 ml/g resin) For manual coupling of a building block, 5 equiv. of Fmoc protected amino acid in DMF (0.5 M), 5 equiv. HBTU in DMF (0.5 M) and 10 equiv. DIPEA (2 M in DMF) were added to the resin. After 2 hours of shaking, the reagent was filtered and the resin was washed. The outcome of the reaction can be monitored with a color test, but mostly a small scale cleavage and subsequent LC-MS analysis on the obtained peptide was preferred.
For manual coupling of the lipid tail, 7 equiv. of the fatty acid, 5 equiv. of HBTU and 10 equiv. of DIPEA were added together and dissolved in 2 mL DMF. The mixture was sonicated and added to the resin. After 2 hours of shaking, the reagent was filtered and the resin was washed. The outcome was monitored with a small scale cleavage and subsequent LC-MS analysis on the obtained peptide.
Intermediate compounds were analyzed by subjecting a small fraction of the peptidyl resin to an acidic cleavage. The released peptide was then analyzed by LC-MS. The following small-scale cleavage conditions with TFA were typically applied: 1 mg of peptidyl resin was brought in a plastic reactor vessel equipped with a Teflon frit. A mixture of TFA/triisopropylsilane/water (95/2.5/2.5 %v/v/v) was added and the vessel was shaken for 30 minutes. The resin was washed with DCM (1 ml) and the combined filtrates were dried using an Argon stream to obtain the crude peptide. Subsequently, the peptides are precipitated in cold MTBE while the protecting group debris remains dissolved. The peptides that dissolved in cold MTBE were precipitated in hexane.
Two different procedures were used for the final cleavage and deprotection step. In the first procedure a solution of TFA/TIS/H2O (95/2.5/2.5 (v/v/v); 10 ml/g resin) was added to the resin and shaken for 15 minutes. The mixture is filtered and the resin is washed 3 times with 1 ml TFA. The filtrate is collected in a Falcon® tube. The cleavage reaction is repeated twice and the combined filtrates were dried (using Ar or N2) to obtain the crude peptide. Thereafter, cold MTBE was added to precipitate the peptidic compounds present in the crude mixture. Precipitation of all peptidic compounds was ensured by 8 minutes of centrifugation (5000 r.p.m). The supernatant, only containing the protecting group debris, was decanted afterwards. This precipitation step was repeated at least three times. The remaining MTBE is evaporated with a gentle Ar-stream.
In the second procedure a solution of 0.1 M HCl in HFIP (equivalent to 1 ml of ca. 37% aq. HCl per 99 ml HFIP) was used to which 1% v/v TIS was added as scavenger. This solution was added to the resin (10 ml/g resin) and reacted for one hour. Then the reaction mixture was filtered and the resin was washed two times with 0.1 M HCl in HFIP. All filtrates were collected in a Falcon® tube. The cleavage reaction and washing steps were repeated three times. After a total time of 4h, the combined filtrates were dried using Ar to obtain the crude peptide. An identical workup procedure with MTBE was followed to remove protecting groups. The removal of all protecting groups was verified using LC-MS analysis. In case of incomplete removal, the crude peptide was treated with the cleavage solution for another 3 to 4 hour and again dried using an Argon stream, followed by precipitation in MTBE.

Production and extraction of natural cyclic lipodepsipeptides
A streak of bacterial cells of Pseudomonas sp. were transferred into 4 × 5 mL King's Broth medium (Difco Laboratories, Sparks, MD, USA) and grown for 1 day under equilibrated conditions (29 °C, 140 rpm shaking frequency). Afterwards, each of the 5 mL cultures were transferred into a 2L Erlenmeyer flask containing 400 mL of KB or M9 minimal medium. Following an additional 24 h cultivation under minimal conditions (28 °C, 150 rpm shaking frequency), the culture was centrifuged to separate the supernatant from the cells.
The supernatant was acidified towards a pH of 2 with use of a 2M HCl solution and was kept at 5°C overnight. Subsequent, the supernatant was once again subjected to centrifugation to separate the collect the precipitated peptide products. With use of a 2M NaOH solution, the pH was set to 8 and the solution was freeze-dried overnight. The resulting crude mixture was dissolved in a minimal amount of methanol prior to purification.
Extraction of the cells was carried out by adding 5ml of an EtOAc/MeOH mixture (50:50) to the cell pellets. The resulting cell solution was subjected to three cycles of freeze-thawing. After subsequent centrifugation, the supernatant was collected and evaporated under high vacuum.
The combined crude fractions obtained from both supernatant and cells were dissolved in MeOH and subjected to RP-HPLC purification.

Extraction and purification of natural CLiP
After growth of P. azadiae SWRI103, the cyclic lipopeptide produced by this bacterial strain was extracted from the supernatant and cell content. In total 5 fractions were caught and examined by mass analysis, with only one showing peptide properties. This peptide fraction was purified via semi-preparative RP-HPLC analysis, using a two solvent system: H2O + 0.1% TFA (A) and CH3CN (B), via application of a linear gradient over 15 minutes going from 25:75 to 0:100 (A:B). The main compound eluted at a retention time of 12.3 min .A total of 6.5 mg of pure peptide was obtained. .

Mass analysis of the natural bananamide analogue
LC-MS analysis confirmed that the main compound found in the crude mixture of the cells was identical to the compound found in the medium extract. HR-MS measurements of the main compound revealed the exact mass of the molecular ion to be 1053.6774 ±0.05Da which agrees to a formula of C52H92N8O14.

NMR characterization of the natural bananamide analogue
The molecular structure of the isolated peptide was revealed by solution state NMR. In acetonitrile-d3, full assignment of the 1 H and 13 C resonances was achieved (see Table I.). The characteristic 2D 1 H-1 H TOCSY patterns revealed the identity of the amino acids ( Figure S5) and the 3-hydroxy decanoic acid fatty acid spin system whereas the 2D 1 H-1 H ROESY spectrum revealed the connectivity of the residues through observation of either H N (i)→ H N (i+1) or H N (i) to H α (i-1) characteristic cross peaks. The carbonyl 13 C resonances and the lactone bond between the hydroxyl functionality of Thr3 and Ile8 could be established by analysing the 1 H-{ 13 C} gHMBC.

Stereochemical investigation of bananamide analogue
To 1.3 mg of peptide, 1.5 ml of a 6M HCl solution was added. The solution was stirred for 22h at a temperature of 90°C, fully hydrolyzing the peptide. Subsequently, the HCl was evaporated with a gentle stream of Ar. The derivatization step was performed by adding 300 µL 1M NaHCO3 and 200µL of a 1% (w/w) solution of 1-fluoro-2-4-dinitrophenyl-5-L-alanine amide (L-FDAA) in acetone to the crude hydrolysis residue. The resulting mixture was let to react for an hour at 40°C. The reaction was quenched by 10µL of a 2M HCl solution. The solution was evaporated with use of a SpeedVac (ThermoFischer). Afterwards, the dried compounds were dissolved in 200µL DMSO of which 20µL was taken out and was diluted to 200 µL with use of a 60:40 ammonium acetate (5mM): acetonitrile buffer prior to injection.
Reversed-phase HPLC analysis of the derivatized hydrolysate was performed on an Agilent Technologies 1260 Infinity II instrument with a Kinetex RP-18 (C18, 150  4.60 mm, 5 µm particle size) column and connected to a multiwavelength detector. The solvents used for the measure of samples are 0.1% TFA in H2O (A) and MeCN (B) at a flow rate of 3 mL min -1 . After sample injection, the column was flushed with 100% A for 3 min, followed by an isothermal (50°C) gradient from 0 to 100 % B over 120 min and subsequent flushing with 100 % B for 3 min. The detector was set to 340 nm to selectively monitor the derivatives.  The Marfey analysis yielded essential information regarding the topology of each individual amino acids, however the presence of D-Leu and L-Leu in a (1:1) ratio impeded to fully assign the absolute configuration of the peptide backbone. It was observed that the discovered peptide consisted out of: D-Ser, D-allo-Thr or D-Thr, L-Ile and D-Glu. The general approach for the synthesis of the three analogues of SWRI103 CLiP (1-3) with different stereochemistry is based on the previously reported synthesis of pseudodesmin A and almost similar procedures were used throughout. (13)(14)(15) A more in-depth discussion about the used design and applicability of the synthetic strategy can be found there. As a similar strategy is used for the synthesis of these three MDN-0066 analogues, its key features are discussed below. Synthesis of (8:6) 1L (1)

Anchoring of Fmoc d-Ser(OH)-OAllyl to a 2-Chlorotrityl chloride resin (7)
Fmoc-D-Ser(OH)-OAllyl (1.189 g, 3.24 mmol, 1.5 equiv.) was dissolved in dry THF (13.5ml) in a dry flask under argon atmosphere and pyridine was added (520 µl, 6.48 mmol, 3 equiv.). The 2-CTC resin (1.60 mmol/g; 1.35 g, 2.16 mmol, 1 equiv.) was brought in a dry reaction vessel (100 ml) and then the first reaction mixture was added to the reactor, which was subsequently flushed with argon and sealed. The reactor was placed in a Selecta Vibromatic shaker and connected to a thermostat with a temperature set at 60°C and the reactor was shaken overnight (24h). Next, the excess of reagents was filtered off, the resin was washed with dry DCM and unreacted functionalities were capped by adding a DCM/MeOH/DIPEA (15 ml; 17/2/1; 210 min.) solution. After washings with DCM (3x), DMF (3x) and DCM (3x), the beads were dried on the oil pump overnight prior to loading determination. The loading was determined by monitoring the dibenzofulvene-piperidine adduct formed after Fmoc-deprotection and a loading of 0.715 mmol/g was obtained.

Automated solid peptide synthesis towards (8)
Automated solid phase peptide synthesis was done on a Syro apparatus. In a C→N fashion, Fmoc-D-Leu-OH, Fmoc-D-Leu-OH, Fmoc-D-allo-Thr-OH, Fmoc-D-Glu-OH and Fmoc-L-Leu-OH were coupled onto the modified 2-CTC resin (100 mg, 0.715mmole). The automated synthesis included the TBS-protected 3-(R)-hydroxydecanoic acid to cap the N-terminus. After the automated synthesis was completed (12h55min), the resin was washed thoroughly with use of THF (3x), DMF (6x) and DCM (3x). Afterwards, the completion of the reaction was checked by a small cleavage test and the obtained peptide was subjected to LC-MS analysis.

Steglich esterification towards (9)
The peptidyl resin was first dried for 2 hours on the oil pump before it was transported to an eppendorf tube (1.5 ml) and stored under argon atmosphere. In a dry flask, Fmoc-L-Ile-OH (227.41 mg; 0.6435 mmol, 10 equiv.) was dissolved in dry THF at 0°C and DIC (90.23 mg, 0.715 mmol, 10 equiv.) was added and, next, this mixture was stirred for 20 minutes at the same temperature. Thereafter, the reaction mixture was added, together with DMAP (1 equiv.) dissolved in dry THF, to the eppendorf tube containing the peptidyl resin. The eppendorf was placed in the thermoshaker and was shaken for 24h at 37°C. Then, the beads were transferred again to the plastic reaction vessel and, subsequently, the resin was washed with THF (3x), DMF (3x) and DCM (3x). A small scale cleavage and LC-MS analysis confirmed ester bond formation.

Coupling of Alloc-L-Leu-OH towards (10)
A stepwise Fmoc deprotection of (9) was performed according to the standard protocol described above. Subsequent, the presence of free amine groups was checked via a standard color test with use of TNBS (2,4,6-trinitrobenzenesulfonic acid). Alloc-L-Leu-OH (77mg; 0.36mmol, 5 equiv.), HBTU (135.59 mg, 0.36mmol, 5 equiv.), DIPEA (100mg, 0.72mmol, 10equiv.) were brought together in a flask and 2ml of DMF was added to dissolve the different compounds. The coupling cocktail was brought to the beats and the mixture was let to shake for 45' at room temperature subsequently, the resin was washed with THF (3x), DMF (3x) and DCM (3x). A small scale cleavage and LC-MS analysis ensured that the coupling was complete.

Allyl & alloc deprotection step and on-resin cyclization towards (1)
Under inert conditions (Ar) while shielded off light, tetrakiss (Pd(PPh3)4) (0.5 equiv., 0,04mmol, 0.042g) was dissolved in a mixture of 1,056ml (60 equiv., 4.29 mmol) PhSiH3 and 4ml dry DMF. The solution was added stepwise to the beads. Firstly, 2ml of the solution was added, letting it react for 1h. Afterwards, the beads were filtered and washed (3x 1ml dry DCM) and another 2ml of the solution was added, repeating the first step. Next, the beads were filtered and the resin was washed thoroughly with 6x DCM, 6x MeOH, 6x DCM and 2x diethyl ether.

Total cleavage and purification of (1)
Total cleavage from the acid-labile 2-CTC resin was done by adding a 0.1M HCl and a 1%TIS in HFIP solution to the peptide reactor. The solution was shaken for 3h and was afterwards filtered in Falcon Tubes® and was blown dry using a gentle argon flow. Precipitation in cold hexane (x2) was done to remove non-peptidic impurities & protecting groups prior to final purification efforts.
Purification was performed using RP-HPLC ( Figure S12). An isothermal gradient of 75%-100% acetonitrile in 15 min was used. All fractions were caught and analyzed. The product was dried overnight at high vacuum. A total of 8mg (11.1% yield, based on resin loading) pure main product was obtained.

Synthesis of (8:6) 4L (2)
The synthesis of the (8:6) 4L proceeded quasi identical as the beforementioned method with the only difference occurring at position 1 & 4 where the configurations of the respective amino acids are altered. To avoid repetition, the previous steps are not discussed and only the final chromatogram of the purification efforts are displayed. A total amount of 5.3 mg was obtained (7.04% yield, based on the initial resin loading). The synthesis of (8:6)-5L variant proceeded very similar as previous analogues. The same peptidyl resin & quantity was used throughout. To avoid repetition, only the conclusive LC-MS analysis of the final purification HPLC chromatogram ( Figure S14) is depicted. A quantity of 6 mg of final peptide was achieved, (7,97% yield, based on initial resin loading).

NMR characterization of the natural orfamide B
The molecular structure of the isolated peptide was revealed by solution state NMR. In DMF-d7, full assignment of the 1 H and 13 C resonances was achieved. The characteristic 2D 1 H-1 H TOCSY patterns revealed the identity of the amino acids and the 3-hydroxy decanoic acid fatty acid spin system whereas the 2D 1 H-1 H ROESY spectrum revealed the connectivity of the residues through observation of either H N (i)→ H N (i+1) or H N (i) to H α (i-1) characteristic cross peaks. The carbonyl 13 C resonances and the lactone bond between the hydroxyl functionality of Thr3 and Ile8 could be established by analyzing the 1 H-{ 13 C} gHMBC.

Stereochemical analysis of the natural CLiPs from Pseudomonas aestus CMR5c
In a 10ml round bottom flask, 0.63 mg orfamide B was dissolved in 1ml 6N aq.HCl and was let to stir for 24h at 100°C.The hydrolysate was dried using a gentle argon flow while simultaneously heating to 60°C. 300 µL of a 1M NaHCO3 solution was added to the residue. White precipitation could be noticed.. Subsequently, 100 µL of a 1%-FDAA in acetone solution was added and it was let to react for 1h at 50°C. A color shift (bright yellow to copper-like orange) could be noticed. The reaction was quenched with 20µL 2M aq.HCl and was dried in vacuo. Afterwards, the dried compounds were dissolved in 200µL DMSO of which 20µL was taken out and was diluted to 200 µL with use of a 60:40 ammonium acetate (5mM): acetonitrile buffer prior to injection. Reversedphase HPLC analysis of the derivatized hydrolysate was performed on an Agilent Technologies 1260 Infinity II instrument with a Kinetex RP-18 (C18, 150  4.60 mm, 5 µm particle size) column and connected to a multiwavelength detector. The solvents used for the measure of samples are 0.1% TFA in H2O (A) and MeCN (B) at a flow rate of 3 mL min -1 . After sample injection, the column was flushed with 100% A for 3 min, followed by an isothermal (50°C) gradient from 0 to 100 % B over 120 min and subsequent rinsing with 100 % B for 3 min. The detector was set to 340 nm to selectively monitor the derivatives.  Synthesis of different stereochemical possibilities of CMR5c-orfamide.
The general approach for the synthesis of the synthetic analogues of orfamide (4-6) with different stereochemistry for the leucine residue at position 1 and 5 in the peptide sequence is based on the previously reported synthesis of pseudodesmin A and almost identical procedures were used throughout (13)(14)(15). A more detailed discussion about the design and development of the synthesis strategy can be found there. The key features of this similar strategy used for the synthesis of these three analogues, are discussed below.

Automated solid peptide synthesis towards (11):
The synthesis of the (10:8) 1L which has the L-leu at position 1 and an D-Leu at position 5 was started from the same preloaded resin (7)  To lower the consumption of the lipid tail, (R) 3-hydroxy decanoic acid, manual coupling was preferred. Note that the main product of CMR5c has a (R) 3-hydroxy tetradecanoic acid as fatty acid moiety. Due to the exceptionally high price of the commercially available (R) 3-hydroxy tetradecanoic acid and the in house availability of (R) 3-hydroxy decanoic acid, we chose to couple this C10 variant. In addition, the usage of another lipid tail analogue does not interfere with our intended purpose as we previously demonstrated that alterations in the length of the lipid tail do not affect the proton-carbon correlation in the characteristic fingerprint CH α region (5).
The success of this elongation of the peptide chain was assessed via a small scale cleavage and of the peptidyl resin (1 mg) and subsequent LC-MS analysis of the obtained peptide fraction. Figure S25: LC-MS analysis of 11 ( Figure S21). The fatty acid tail was coupled manually.

LC-MS analysis
[M+H] + was 1183.73 Da which was in agreement with C57H101N9O1.

Steglich esterification towards (12)
The formation of the esterbond was performed using Alloc-L-Ile as a building block. First, the peptidyl resin was dried via several washing steps with Et2O (3x) and further dried at the oil pump when necessary. Thereafter, the resin beads were transferred to an Eppendorf tube (1.5 ml) and stored under argon atmosphere. In a dry flask, Alloc-L-Ile-OH (0.154 g; 0.716 mmol, 10 equiv.) was dissolved in dry THF at 0°C and DIC (99.5 mg, 0.788 mmol, 11 equiv.) was added. The mixture was stirred for 20 minutes at the 0°C. DMAP (1 equiv.) dissolved in dry THF. Both solutions were added to the Eppendorf tube containing the peptidyl resin. The Eppendorf was sealed with parafilm, placed in a thermoshaker and shaken for 24h at 37°C. Then, the beads were transferred back to the plastic reaction vessel and, subsequently, the resin was washed with THF (3x), DMF (6x), DCM (6x) and again DMF (6x). A small scale cleavage and LC-MS analysis confirmed ester bond formation. Figure S26: LC-MS analysis of 12 ( Figure S21).
[M+H] + was 1326.8 Da which was in agreement with C63H110N10O20.

Allyl/Alloc deprotection and on-resin cyclization (4)
The dry resin beads were first washed with dry DCM under inert conditions and after 2 mL of dry DCM was added to the reaction vessel. Phenylsilane (4.29 mmol, 60 equiv.) and a catalytic amount of Pd(PPh3)4 (0.018 mmol, 0.25 equiv.) were also added. Next, the mixture with the peptidyl resin was flushed with Argon and shielded from light. After the reaction vessel was shaken for 1 hour, the solution was filtered, the resin was washed with dry DCM (3x) and the deprotection step, described above, was repeated once more. Thereafter, the resin wash thoroughly washed with DMF (6x), DCM (6x), MeOH (3x) and DMF (6x) and dried by washing with Et2O (3x).
For the on-resin cyclization, HATU (5 equiv.) and HOBt (5 equiv.) were added together and dissolved in 2 mL dry DMF. After DIPEA (5 equiv.) was added to the solution, the solution was sonicated and added to the resin under argon atmosphere. The reaction vessel was shaken for 4 hours followed by filtration and washing of the resin with DMF (6x), DCM (6x), DMF (6x). Successful deprotection and cyclization was confirmed via LC-MS analysis of the peptide fraction (4) obtained after a small scale cleavage.

Total deprotection and purification (4)
Total deprotection and cleavage from the acid-labile resin was performed using a solution of 0.1M HCl in HFIP (equivalent to 1 mL of ca. 37% HCl per 99 mL HFIP) to which 1% of scavenger, TIS, was added. After addition of 4 mL of this cleavage solution to the peptidyl resin (0.0716 mmol), the reaction vessel was shaken for 1 hour. The mixture was filtered and the resin was washed with DCM. This cleavage from the resin was repeated 2 more times. The combined filtrates were collected in a falcon tube and dried using an argon stream to obtain the crude peptide. To avoid incomplete deprotection, the dried fraction in the falcon tube was dissolved in the cleavage solution and reacted for another 4 hours. The volatile solution was evaporated using argon and the solid remainder with the crude peptide was precipitated in cold MTBE. After, the MTBE solution was centrifuged for 8 minutes at 5°C, the solution containing the protecting groups was decanted and the precipitation step was repeated 2 more times. The crude peptide 4 was obtained as a whitishyellow precipitate.

Synthesis of (10:8) 5L (5)
The synthesis of the (10:8) analogue with a L-leucine at position 5 and an D-leucine at position 1, proceeded according to the same synthesis strategy described above for (10:8) 1D. The synthesis was started from the preloaded resin 7 (0.358 mmol/g; 200 mg; 0.0716 mmol) and all building blocks were available. The synthesis proceeded under identical conditions as mentioned above. Although the crude purity seems quite low, LC-MS analysis confirmed the successful synthesis of (10:8) 5L (5) after total deprotection and cleavage from the resin using the standard procedure with 0.1M HCl in HFIP and MTBE work-up.   The standard procedure for final cleavage and total deprotection using 0.1M HCl in HFIP was applied, followed by work-up with MTBE. The crude peptide 6 was obtained as a whitish powder and purified using the same gradient and solvent system as for (10:8) 5D. The overall yield starting from the initial resin loading is 5.11 %.

Extraction and purification of natural CLiP
After growth of P. sp. PH1b, the cyclic lipopeptides produced by this bacterial strain was extracted from the supernatant and cell content. In total 8 fractions were collected and examined by mass analysis. The main compound eluted at a retention time of 20.6 min in Fig S1. This peptide fraction was purified via semi-preparative RP-HPLC analysis, using a     Consecutively, the mixture was dried using a gentle N2 stream while keeping the temperature at 60°C to enhance evaporation. Next, 100µL of a 1M NaHCO3 solution was added to the dried, crude mixture to quench the reaction. Subsequently, 150µL of a 1% (m/m%) solution of Marfey's reagent (1-fluoro-2-4-dinitrophenyl-5-L-alanine amide) in HPLC grade acetone (VWR) was added. The solution was transferred to an Eppendorf and was let to react in a Thermoshaker (1100 r.p.m, 40°C) for approximately an hour. Thereafter, 50µL of a 2M HCl solution was used to quench the nucleophilic aromatic substitution reaction and the solution was dried using a SpeedVac (Thermofisher). The resulting dried substance was redissolved in 200µL DMSO of which 30 µL was taken out to prepare an HPLC sample. The 30µL was diluted using a mixture (40/60acetonitrile/5mM ammonium acetate). The solvents used for the measure of samples are 0.1% TFA in H2O (A) and MeCN (B) at a flow rate of 3 mL min -1 . After sample injection, the column was flushed with 100% A for 3 min, followed by an isothermal (50°C) gradient from 0 to 100 % B over 120 min and subsequent flushing with 100 % B for 3 min. The detector was set to 340 nm to selectively monitor the derivatives.

Extraction and purification of natural CLiP
Extraction and purification of xantholysin A was performed as previously reported (8).

Stereochemical analysis of xantholysin A, excreted by P. mosselii BW11M1
1.3 mg of xantholysin A was dissolved in 10ml 6N HCl and the solution was let to react for 24h at 95°C. Afterwards the crude mixture was dried using a gentle Ar-flow while simultaneously applying heat (50°C) to enchance evaporation. 200µL of a 1M NaHCO3 solution was added and 360 µL of a 1% FDAA in acetone solution was added. The derivatization step was let to proceed during one hour (40°C). Quenching was done using 80µL of a 2M HCl solution and was let to dry. The resulting dried substance was redissolved in 200µL DMSO of which 30 µL was taken out to prepare an HPLC sample. The 30µL was diluted using a mixture (40/60-acetonitrile/5mM Ammoniumacetate). The solvents used for the measure of samples are 0.1% TFA in H2O (A) and MeCN (B) at a flow rate of 3 mL min -1 . After sample injection, the column was flushed with 100% A for 3 min, followed by an isothermal (50°C) gradient from 0 to 100 % B over 120 min and subsequent flushing with 100 % B for 3 min. The detector was set to 340 nm to selectively monitor the derivatives.

Multidisciplinary data of xantholysin A from P. mosselii BW11M1
We first retrieved the previously available data from both the bioinformatic and the chemical analysis workflows from  (Table 3). Consequently, there is a mismatch between the prediction and Marfey's analysis both for the leucines and for the glutamines/glutamic acid. Nevertheless, the occurrence of L CL domains in module 12 and 13 of the NRPS assembly line allow to designate Leu11 and Leu12 as L-configured, reducing the positional ambiguity of the remaining L-Leu and both D-Leu to three possibilities. As for the five Glx residues, five different distributions of the only L-Glx residue are possible. Since the positional ambiguity of the leucines is independent from that of the Glx residues, 15 different sequences should be considered at this point. Given the considerable expenditure required to synthesize 15 different sequences, prioritization is in order based on all available information. Considering the proposed structural similarity between xantholysin A and MA026, it appeared evident to synthesize this compound using the same total synthesis approach applied for bananamide and orfamide, as described in detail in the supplementary materials section. As is clearly demonstrated by the superposition of the (C-H) spectral fingerprint of the purified synthetic (   Fmoc-L-Leu were coupled onto the Rink Amide resin (50 mg, 0.69mmole/g). The automated synthesis included the TBS-protected 3-(R)-hydroxydecanoic acid to cap the N-terminus. After the automated synthesis was completed, the resin was washed thoroughly with use of THF (3x), DMF (6x) and DCM (3x). Afterwards, the completion of the reaction was checked by a small cleavage test and the obtained peptide was subjected to LC-MS analysis.

Esterfication with Alloc-L-Ile towards (14)
The Steglich esterification was carried out by treating the peptidyl resin with 5eq. of Alloc-L-Ile-OH, 5 eq. pyridine and 0.20 eq. DMAP in dry DMF. The mixture was let to shake for 2x 24h at room temperature. The beads were extensively washed with 8x DMF, 8x DCM and 4x Et2O. The reaction completion was verified using a test cleavage.
The cyclized peptides were cleaved off by using a cleavage cocktail consisting of 0.1M HCl in HFIP +1% TIS. The cleavage reaction was let to shake for 5 hours, ensuring full deprotection of the peptide. The obtained crude mixture was analyzed using LC-MS (S33). Purification of (15): The crude mixture was dissolved in a minimal amount of methanol prior to subjecting to HPLC purification.This peptide fraction was purified via semi-preparative RP-HPLC analysis ( Figure S56

Quantifying the (dis)similarity in NMR fingerprints
Using a suitable metric, one could quantify the spectral similarity between two fingerprints. Typically, the difference in 1 H (Δ 1 H) or 13 C shifts (Δ 13 C) can be used. In some cases, the 13 C chemical shift difference for a particular α(C-H) is modest while that of its 1 H is quite distinct. Therefore, we find that combined use of 1 H and 13 C chemical shifts provides the best differentiation. To resort to a single value per residue, we use the Euclidian chemical shift distance d = SQRT[(  H) 2 +( 13 C) 2 ] between two peaks to be matched in the 1 H-13 C HSQC fingerprint. (Applied to the case of bananamide SWRI103 ( Figure S62A) and orfamide CMR5c ( Figure S62B), it supports the attribution of stereochemistry in a more quantitative way.